RESUMO

Many patients under therapy with recombinant human erythropoietin (rhuEPO) show resistance to the treatment, an effect likely associated with the accumulation of tissue factors, especially in renal and cardiovascular diseases. Hyperhomocysteinemia due to high serum levels of homocysteine has been suggested among the risk factors in those pathologies. Its main effect is the N-homocysteinylation of proteins due to the interaction between the highly reactive homocysteine thiolactone (HTL) and lysine residues. The aim of this study was to evaluate the effect of N-homocysteinylation on the erythropoietic and antiapoptotic abilities of EPO, which can be a consequence of structural changes in the modified protein. We found that both cellular functions were altered in the presence of HTL-EPO. A decreased net positive charge of HTL-EPO was detected by capillary zone electrophoresis, while analysis of polyacrylamide gel electropherograms suggested formation of aggregates. Far-UV spectra, obtained by Circular Dichroism Spectroscopy, indicated a switch of the protein's secondary structure from α-helix to ß-sheet structures. Results of Congo red and Thioflavin T assays confirm the formation of repetitive ß-sheet structures, which may account for aggregates. Accordingly, Dynamic Light Scattering analysis showed a markedly larger radius of the HTL-EPO structures, supporting the formation of soluble oligomers. These structural changes might interfere with the conformational adaptations necessary for efficient ligand-receptor interaction, thus affecting the proliferative and antiapoptotic functions of EPO. The present findings may contribute to explain the resistance exhibited by patients with cardio-renal syndrome to treatment with rhuEPO, as a consequence of structural modifications due to protein N-homocysteinylation.

Assuntos

Apoptose , Proliferação de Células , Eritropoetina/química , Homocisteína/análogos & derivados , Lisina/química , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Células Cultivadas , Eritropoetina/metabolismo , Homocisteína/química , Humanos , Megacariócitos/metabolismo , Megacariócitos/patologia , Ligação Proteica

Assuntos

Eritropoetina/análogos & derivados , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Ativação Enzimática , Eritropoetina/genética , Eritropoetina/metabolismo , Eritropoetina/farmacologia , Humanos , Janus Quinase 2/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Receptores da Eritropoetina/metabolismo

RESUMO

It is now recognized that in addition to its activity upon erythroid progenitor cells, erythropoietin (Epo) is capable of stimulating survival of different non-erythroid cells. Since stimulation of erythropoiesis is unwanted for neuroprotection, Epo-like compounds with a more selective action are under investigation. Although the carbamylated derivative of erythropoietin (cEpo) has demonstrated non-hematopoietic tissue protection without erythropoietic effect, little is known about differential mechanisms between Epo and cEpo. Therefore, we investigated signaling pathways which play a key role in Epo-induced proliferation. Here we show that cEpo blocked FOXO3a phosphorylation, allowing expression of downstream target p27(kip1) in UT-7 and TF-1 cells capable of erythroid differentiation. This is consistent with the involvement of cEpo in slowing down G1-to-S-phase progression compared with the effect of Epo upon cell cycle. In contrast, similar antiapoptotic actions of cEpo and Epo were observed in neuronal SH-SY5Y cells. Inhibition and competition assays suggest that Epo may act through both, the homodimeric (EpoR/EpoR) and the heterodimeric (EpoR/ßcR) receptors in neuronal SH-SY5Y cells and probably in the TF-1 cell type as well. Results also indicate that cEpo needs both the EpoR and ßcR subunits to prevent apoptosis of neuronal cells. Based on evidence suggesting that cell proliferation pathways were involved in the differential effect of Epo and cEpo, we went forward to studying downstream signals. Here we provide the first evidence that unlike Epo, cEpo failed to induce FOXO3a inactivation and subsequent p27(kip1) downregulation, which is clearly shown in the incapacity of cEpo to induce erythroid cell growth.

Assuntos

Eritropoetina/análogos & derivados , Eritropoetina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Eritropoese/genética , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fase G1/efeitos dos fármacos , Fase G1/genética , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Transdução de Sinais/genética